The present invention relates to a method of eliminating electrophoretic rate differentials in the analysis of nucleotides.
In the prior art, it was necessary to subject a DNA fragment (genetic material) to complex chemical and physical testing in order to determine the "spelling" of a genetic "word". Typically, such techniques involved the use of an electron microscope or X-ray difraction in order to, often quite laboriously, determine which of the four basic nucleotides were present at each of many thousands of possible locations.
Recently, the prior art has seen the usage of electrophoresis techniques applied to acrylimite gels embedded with genetic material to bring about separations of the nucleic acids within the DNA molecule. Electrophoresis techniques involve the usage of considerable voltage in order to force a migration of the nucleic acid through the acrylimite gel, this being under the influence of both the voltage and the gravity vector.
A problem in such prior art techniques, one example of which exists in U.S. Pat. No. 4,224,134 to Hoefer, was that the heat generated by the necessary voltage often operated to degrade both the integrity of the test material and to reduce the readability of the test results in that the acrylimite gel is ideally kept at about 53.degree. Centigrade and, thereby, is structurally vulnerable to excursions of heat and temperature.
In addition, the methodology of holding the gel during the period of testing and thereafter has contributed to the problem of structural and chemical integrity of the gel, and therefore the reliability of the test results.
Prior art attempts to deal with the above problem of heat and temperature excursions have related to reducing the voltage used in the electrophoresis process; however, as voltage is reduced, the velocity at which the DNA material will travel through the vertical gel is reduced, thereby considerably increasing the time required for a given study and, in addition, often reducing the number of letters within a DNA sequence that can be accurately measured. Accordingly, the prior art has been aware that a maximum voltage applied to the acrylimite gel is desirable so long as the temperature of the gel can be controlled. However, prior efforts to achieve this result have not been satisfactory. Therefore, it is to this problem that the present invention is addressed.
Other relevant prior art of which the Applicants are aware includes U.S. Pat. Nos. 3,677,930 (1972) to Meshbane; 3,980,540 (1976) to Hoeffer; 4,305,799 (1981) to Schwartz; and 4,142,960 to Hahn. The publication of E. Heftmann, entitled Chromotography, 2nd Edition, Reinhold Publishing Co., New York (1967) pp. 235 to 237 sets forth the theory of high voltage electrophoresis.